Influenza sensor

ABSTRACT

A sensor for the detection of tetrameric multivalent neuraminidase within a sample is disclosed, where a positive detection indicates the presence of a target virus within the sample. Also disclosed is a trifunctional composition of matter including a trifunctional linker moiety with groups bonded thereto including (a) an alkyl chain adapted for attachment to a substrate, (b) a fluorescent moiety capable of generating a fluorescent signal, and (c) a recognition moiety having a spacer group of a defined length thereon, the recognition moiety capable of binding with tetrameric multivalent neuraminidase.

BENEFIT OF PRIOR APPLICATION

[0001] This application claims the benefit of the filing date of U.S.Provisional Application No. 60/162,427, filed Oct. 28, 1999

FIELD OF THE INVENTION

[0002] The present invention relates to a diagnostic sensor for thedetection of influenza virus and to a method of detecting influenzavirus with such a diagnostic sensor. This invention was made withgovernment support under Contract No. W-7405-ENG-36 awarded by the U.S.Department of Energy. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

[0003] The early diagnosis of influenza infection is important forseveral reasons. One reason is that it is critical to be able to rapidlyscreen influenza from other infectious diseases in the event of abio-agent attack. Most scenarios for bio-agent attacks show a slowedresponse to the recognition that an attack has taken place primarilybecause diseases such as anthrax and smallpox present flu-like symptoms.Medical personnel do not have a rapid and simple screen for influenzainfection and, consequently, victims can be miss-diagnosed as having theflu and sent home. A delay of even a few days in the recognition of abio-agent attack can have adverse affect on the minimization of theimpact of an attack.

[0004] Another reason for a rapid diagnostic for influenza is importantis in helping to avert a worldwide pandemic in the event that a newstrain like the 1918 swine flu appears. Rapid screening with inexpensivefieldable sensors is essential to rapidly pinpoint a new potentialoutbreak. Although it is also important to specify the strain of theinfluenza infection, it is first critical to rapidly identify anoutbreak and this can only be done using a flexible, inexpensive,fieldable sensor.

[0005] Recently, a number of high binding affinity neuraminidase (alsoknown as sialidase) inhibitors have been developed and shown to be quiteeffective in curing the flu but only if such inhibitors are administeredearly on in the infection (generally within the first 24 to 48 hours).At present, these drugs can not be effectively used as there is not asimple diagnostic tool that can be used to detect the influenza virusearly enough to effectively use neuraminidase inhibitors. The onlytechnologies currently capable of early diagnosis of influenza arelab-based approaches like ELISA, which are instrument and personnelintensive, expensive, and slow. What is needed is a simple inexpensivediagnosis that can be easily used in either a clinical or field settingand yet have at least the same specificity and sensitivity as ELISA.Accordingly, it is highly desirable to develop a rapid diagnosis forinfluenza to facilitate the treatment of influenza using suchneuraminidase inhibitors.

[0006] An optical biosensor system has recently been developed torapidly detect protein toxins, e.g., cholera, shiga, and ricin (see,U.S. patent application Ser. No. 09/338,457, by Song et al., filed Jun.22, 1999). The integrated optical biosensor developed for the detectionof protein toxins was based on proximity-based fluorescence changes thatare triggered by protein-receptor binding. In demonstrations of thisapproach for the detection of cholera and avidin using flow cytometry,it was shown that this technique was as sensitive as ELISA. In contrastto ELISA, such an optical biosensor can be much faster (minutes),simpler (a single step with no added reagents) and robust owing to thestability of the recognition molecules (glycolipids and biotin) andmembranes. More recently, an optical biosensor system has beenincorporated into planar optical waveguides (see, U.S. ProvisionalPatent Application Serial No. 60/140,718, by Kelly et al., filed Jun.22, 1999) and shown to have sensitivity equivalent to that of flowcytometry. The demonstration of such an optical biosensor using planaroptical waveguides provides a path towards the development ofminiaturized sensor arrays.

[0007] One object of the present invention is adaptation of such abiosensor to sensing applications directed to the detection of influenzavirus.

[0008] Another object of the present invention is the selection andchemical modification of receptors that bind neuraminidase and thatallow attachment of such receptors to membranes together with theincorporation of fluorophores.

SUMMARY OF THE INVENTION

[0009] To achieve the foregoing and other objects, and in accordancewith the purposes of the present invention, as embodied and broadlydescribed herein, the present invention provides for the detection oftetrameric neuraminidase within a sample, where a positive detectionindicates the presence of a target virus within said sample, said sensorincluding a surface, recognition molecules situated movably at saidsurface, said recognition molecules capable of binding with saidtetrameric multivalent neuraminidase, said recognition molecules furthercharacterized as including a fluorescence label thereon, and, a meansfor measuring a change in fluorescent properties in response to bindingbetween multiple recognition molecules and said tetramericneuraminidase.

[0010] The present invention further provides a method of method ofdetecting tetrameric neuraminidase within a sample, where a positivedetection indicates the presence of a target virus within said sample,said method including contacting a sample with a sensor including asurface, recognition molecules situated movably upon said surface, saidrecognition molecules capable of binding with said tetramericmultivalent neuraminidase wherein said recognition molecules include afluorescence label thereon, and measuring a change in fluorescentproperties in response to binding between multiple recognition moleculesand said tetrameric neuraminidase.

[0011] The present invention further provides for the detection oftetrameric neuraminidase within a sample, where a positive detectionindicates the presence of a target virus within said sample, said sensorincluding a surface, at least two different recognition moleculessituated movably upon said surface, said recognition molecules capableof binding with said tetrameric multivalent neuraminidase wherein atleast one recognition molecule includes a fluorescence donor labelthereon and at least one recognition molecule includes a fluorescenceacceptor label thereon, and, a means for measuring a change influorescent properties in response to binding between at least twodifferent multiple recognition molecules and said tetramericneuraminidase.

[0012] The present invention further provides a method of method ofdetecting tetrameric neuraminidase within a sample, where a positivedetection indicates the presence of a target virus within said sample,said method including contacting a sample with a sensor including asurface, at least two different recognition molecules situated movablyupon said surface, said recognition molecules capable of binding withsaid tetrameric multivalent neuraminidase wherein at least onerecognition molecule includes a fluorescence donor label thereon and atleast one recognition molecule includes a fluorescence acceptor labelthereon, and measuring a change in fluorescent properties in response tobinding between multiple recognition molecules and said tetramericneuraminidase.

[0013] The present invention still further provides a trifunctionalcomposition of matter including a trifunctional linker moiety includingas groups bonded thereto (a) an alkyl chain adapted for attachment to asubstrate, (b) a fluorescent moiety capable of generating a fluorescentsignal, and (c) a recognition moiety having a spacer group of a definedlength thereon, said recognition moiety capable of binding withtetrameric multivalent neuraminidase.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 shows a diagram of an exemplary receptor molecule includingthe three necessary functionalites.

[0015]FIG. 2 shows a diagram of another exemplary receptor moleculeincluding the three necessary functionalites.

DETAILED DESCRIPTION

[0016] The present invention concerns a diagnostic sensor for thedetection of influenza virus and to a method of detecting influenzavirus with such a diagnostic sensor. In particular, the presentinvention concerns a diagnostic sensor capable of detecting organismssuch as influenza virus that contain neuraminidase.

[0017] Organisms that contain neuraminidase include bacteria (Vibriocholerae, Clostridium perfringens, Streptococcus pneumoniae, andArthrobacter sialophilus) and viruses (influenza virus, parainfluenzavirus, mumps virus, Newcastle disease virus, fowl plague virus, andsendai virus). In viruses, neuraminidase occurs as a tetramer. Thistetrameric structure facilitates the operation of the sensor of thepresent invention. Detection of neuraminidase activity related to anytetrameric neuraminidase structure in a virus is within the scope of thepresent invention. Detection of neuraminidase from influenza virus isparticularly desired.

[0018] The selection of receptors is related to the prior work that hasbeen done to synthesize neuraminidase inhibitors having high bindingaffinities. In principal, any neuraminidase inhibitor could be used butpreferably the present influenza sensor will incorporate thoseneuraminidase inhibitors that have the highest binding affinities.Typically, neuraminidase inhibitor compositions having in vitro K_(i)(inhibitory constants) of less than about 5×10⁻⁶M, typically less thanabout 5×10⁻⁷M and preferably less than about 5×10⁻⁸M are excellentcandidates for the recognition portion of the influenza sensor of thepresent invention.

[0019] In one embodiment, the influenza sensor of the present inventioninvolves: 1) formation of a biomimetic membrane, which incorporatesfluorescent dye-labeled receptors for neuraminidase, on the surface ofan optical transducer (this could be a glass bead for flowcytometry—FCM—or a planar optical waveguide for an integrated opticalbiosensor); 2) the chemical modification of selected neuraminidaseinhibitors to attach them to a membrane and to also attach fluorophoresthat fold into the fluid upper leaf of the membrane; 3) the detection,using a planar optical waveguide of a microsensor array or FCM, ofenvelope proteins or the influenza viral particle directly as measuredby a shift in the ratios of intensity of two individualfluorescently-emitted signals, and 4) the use of multiple receptors inthe recognition effect (in influenza neuraminidase is a tetramer) toinsure extremely high effective binding affinities (avidity) and, as aresult, ultrahigh sensitivities and specificities.

[0020] One advantage of the present invention is the ultrahighsensitivity and specificity obtained by using multiple receptors eachwith high binding affinities (the avidity effect) to help insure earlydiagnosis, e.g., within the first 12 hours after infection. Anotheradvantage of the present invention is the simplicity and speed ofoperation which makes detection fast and operation possible in a varietyof situations. Another advantage of the present invention is theelimination of the need for additional reagents or additives therebysimplifying the use and extending shelf storage lifetimes. Anotheradvantage of the present invention is the flexibility in adaptation toeither flow cytometry or to miniaturized sensor systems utilizing planaroptical waveguide permits use in a variety of clinical or fieldsituations. Another advantage of the present invention is the robustnessof the sensor system that results from the high stability of thereceptor molecules and the active membrane. Another advantage of thepresent invention is the simplicity of sample introduction whichminimizes front-end sample preparation.

[0021] Coupling recognition to signal transduction and amplification canbe conducted as follows. The sensor of the present invention mimics manycell signaling processes in nature by directly coupling a recognitionevent to signal transduction and amplification. In the case of thepresent influenza sensor, the sensor relies on recognition of chemicallymodified sialic acid-like receptors by neuraminidase, an envelopeprotein for influenza. As neuraminidase in a virus such as influenza isa multivalent protein (neuraminidase is a tetramer), binding will bringseveral receptors into close proximity thereby triggering proximitybased fluorescence changes. The selection of target receptors isdiscussed below. The receptor molecules are chemically modified to bothattach fluorescent tags and to attach them to the fluid upper leaf of aphospholipid bilayer. The choice of a phospholipid bilayer is importantfor several reasons. First, this is an excellent mimic of a cellmembrane surface, which is the natural target of envelope viruses.Second, the use of membrane mimics helps minimize non-specific proteinabsorption and the attendant nonspecific response of the sensor element.Third, the upper leaf of the membrane is fluid thereby insuring that thereceptor molecules and their fluorescent tags are mobile and, if theconcentration is low, relatively distant thereby minimizing proximitybased fluorescence changes prior to protein binding. The binding eventbetween neuraminidase and the sialic acid-like receptors then bringsmultiple receptors into close proximity triggering the fluorescencechanges. There are two proximity based fluorescence changes that can beused for detection. The simplest is self-quenching of fluorescence thatresults in a sharp decrease in the fluorescence of the fluorophoreattached to the receptor molecules. The second is resonant energytransfer (FRET) where donor fluorophores transfer their energy to theacceptor fluorophores that are attached to the receptor molecules. Inthis case, the receptor molecules are tagged with both fluorescentdonors and acceptors (typically in a 1:1 ratio). FRET results in a colorchange in the fluorescence, which can be more easily distinguished fromsimple self-quenching in terms of being directly coupled to thereceptor-protein recognition. Preferably, the selection of fluorophoresis such that there must be overlap of the donor emission with theexcitation profile of the acceptor. Many pairs of fluorophores exhibitthis relationship and the selection is primarily dictated by thestability of the fluorophores and the most effective separation of theemission profiles to minimize background fluorescence.

[0022] Influenza is an RNA virus and, therefore, has a rapid antigenicdrift and antigenic shift. As a result, the binding of antibodies andreceptor molecules to neuraminidase is constantly changing even within aparticular strain. However, antigenic shift and drift do not affectbinding of a neuraminidase inhibitor which bind to silacic acid. Forthis reason, the present invention has targeted the binding region ofneuraminidase that targets sialic acid residues on the cell membranesurface. It is through neuraminidase binding to sialic acid that theinfluenza virus particle invades the host cell through membrane fusion.As this binding event is critical to viral particle invasion, it islikely that the binding pocket in neuraminidase that selects sialic acidis relatively invariant. There have been several crystal structures ofneuraminidase measured with differing inhibitors (molecules that mimicsialic acid but which have even higher binding affinities toneuraminidase) that show the binding pocket for sialic acid site isrelatively invariant. Moreover, there has been a great deal of work insynthesizing neuraminidase inhibitors that have exceptionally highbinding affinities and these molecules are all good potential receptorsfor the influenza sensor of the present invention. As noted below,initial selection was of a few molecules that have binding affinities inthe micromolar range. The use of molecules that target the sialic acidbinding site insures that this sensor will be effective over a longperiod of time and to virtually any strain of influenza. Moreover, asneuraminidase is multivalent with regard to binding sialic acid, the useof inhibitors and sialic acid variants insures that the above sensortransduction scheme (proximity based fluorescence changes) can work and,equally important, that the effective binding affinities are high byvirtue of the avidity effect.

[0023] Chemical modification of the neuraminidase receptors can be asfollows. A number of neuraminidase inhibitors derived from 3,4-diaminobenzoic acid have been described in the literature. These compounds bindto the sialic acid binding site on neuraminidase. One of these3,4-diamino benzoic acid-based neuraminidase inhibitors,4-acetylamino-3-guanidine benzoic acid is reported to bind NA with anaffinity constant of 10⁵. The present approach to a neuraminidasedetector involves covalently linking this inhibitor to a fluorescentmolecule that is anchored into a membrane. The covalent attachment isaccomplished as follows. 3,4-Diamino benzoic acid (I) is acetylated onthe 4-amino group to yield 3-amino-4-acetylamino benzoic acid (II).Compound II is then alkylated to yield 3-alkylamino-4-acetylaminobenzoic acid (III). Treatment of III with cyanogen bromide followed byammonia yield compound IV that may serve as a neuraminidase receptor inthe present invention.

[0024] The neuraminidase receptor (IV) can be linked to the fluorescentprobe a number of ways. One possible approach is as follows. Firstcompound IV is attached to a polyethylene glycol spacer to thealphα-amino group of lysine. The alpha-carboxyate is modified as anamide to a 16, 17, or 18-carbon hydrocarbon, which serves as a membraneanchor. The e-amino group of lysine is modified to carry a hydrophobicfluorescent molecule such as a Bodipy molecule.

[0025] Preparation of the Membrane Architectures on Optical Transducerscan be as follows. As noted above, the receptor molecules would havebeen chemically modified to bind them into the upper leaf of aphopholipid bilayer through attachment to two aliphatic side chains.Moreover, the fluorophore has been selected and attached in a way thatinsures that it folds over into the upper leaf of the bilayer. The factthat the fluorophore resides in the upper leaf of the bilayer isimportant for two reasons. First, this insures that the fluorophoreitself and the linker that attaches it to the lipid tail do notinterfere with recognition by providing a non-specific site for proteinbinding. Second, the residence of the fluorophore in the upper leafprovides additional stability of the receptor-membrane structure. Themembranes are then fabricated onto optical transducers (either glassbeads for FCM or planar optical waveguides for microsensor arrays) byvesicle fusion onto hydrophobic or hydrophilic surfaces. The simplestapproach is vesicle fusion onto a hydrophilic surface to form a supportbilayer. This can be done in a flow cell where the surface can beexposed for a period of time (hours) to a solution containing thevesicles. The second approach is to spread vesicles containing thetagged receptors over a methyl terminated self-assembled monolayer toform a hybrid bilayer where the lower leaf is covalently attached to thetransducer surface. The hybrid bilayer has the advantage of betterlong-term stability.

[0026] In the present invention, the architecture of the fluid membranescan be as a regular bilayer membrane where both layers are depositedupon a support surface, can be a hybrid bilayer, e.g., where a firstlayer is covalently attached to an oxide surface, can be a selectivelytethered bilayer on an oxide surface, where a membrane molecule iscovalently bonded to the oxide substrate, or a bilayer cushioned by apolymer film. Supported membranes useful in the practice of the presentinvention are generally described by Sackmann, in “Supported Membranes:Scientific and Practical Applications”, Science, vol. 271, no. 5245, pp.43-45, Jan. 5, 1996. Hybrid bilayer membranes or selectively tetheredbilayer membranes may be more preferred as such membranes may havegreater stability over time and therefor provide greater shelf lifetimesfor sensor applications. Additionally, a surface with mobile receptorssuch as an oxide surface with mobile receptor molecules thereon canserve as a platform in the present invention.

[0027] Bilayer membranes can be formed upon a planar oxide substrate,e.g., by initially forming vesicles followed by vesicle fusion orspreading of, e.g., phospholipid, bilayers on glass substrates as iswell known to those skilled in the art.

[0028] In one embodiment of the present invention, the transductionelement used is fluorescein, which has a high extinction coefficient, ahigh fluorescence quantum yield and proximity-dependent fluorescenceself-quenching. Other suitable fluorescent dyes are well known to thoseof skill in the art. Fluorescein may be covalently attached to a freefunctional group by appropriate coupling to produce afluorescein-labeled moiety. The fluorescein should have minimalinfluence on the binding affinity of the recognition portion of thefinal molecule to the influenza virus.

[0029] In another embodiment of the present influenza sensor, thesensing molecules can be functionalized with either an acceptor dyemolecule or a donor dye molecule whose excitation spectra overlap forefficient energy transfer. In effect, excitation of a blue emitting dyecan result in fluorescence with a maximum at roughly 570 nm whenfunctionalized Bodipy is free to move about in the bilayer membrane.Upon exposure to influenza virus (tetrameric neuraminidase), both thedonor and the acceptor dyes are brought into close proximity. This canresult in an energy transfer and a decrease in the fluorescence at 570nm and a concomitant increase in the fluorescence of the acceptor dyethat has its fluorescence maximum at roughly 630 nm. Such a simultaneousincrease in the red fluorescence and decrease in the blue fluorescenceis a highly distinguishing feature of this sensor approach. In effect, atwo-color fluorescence measurement can be used to probe the intensity offluorescence from both dye molecules. Only a specific binding eventbetween the neuraminidase and the receptor will give rise to such asimultaneous increase in one fluorescent signal with a decrease in theother. Any change in the environment will give rise to shifts of thefluorescence of both dye molecules. Such an energy transfer approachprovides a means for self-referencing in such sensor applications.

[0030] A number of exemplary methods for the preparation of thecompositions of the invention are provided below. These methods areintended to illustrate the nature of such preparations are not intendedto limit the scope of applicable methods.

[0031] Generally, the reaction conditions such as temperature, reactiontime, solvents, workup procedures, and the like, will be those common inthe art for the particular reaction to be performed. The cited referencematerial, together with material cited therein, contains detaileddescriptions of such conditions. Typically the temperatures will be−100° C. to 200° C., solvents will be aprotic or protic, and reactiontimes will be 10 seconds to 10 days. Workup typically consists ofquenching any unreacted reagents followed by partition between awater/organic layer system (extraction) and separating the layercontaining the product.

[0032] Oxidation and reduction reactions are typically carried out attemperatures near room temperature (about 20° C.), although for metalhydride reductions frequently the temperature is reduced to 0° C. to−100° C., solvents are typically aprotic for reductions and may beeither protic or aprotic for oxidations. Reaction times are adjusted toachieve desired conversions.

[0033] Condensation reactions are typically carried out at temperaturesnear room temperature, although for non-equilibrating, kineticallycontrolled condensations reduced temperatures (0C to −100° C.) are alsocommon. Solvents can be either protic (common in equilibratingreactions) or aprotic (common in kinetically controlled reactions).

[0034] Standard synthetic techniques such as azeotropic removal ofreaction by-products and use of anhydrous reaction conditions (e.g.inert gas environments) are common in the art and will be applied whenapplicable.

[0035] As discussed in this application, in a preferred embodiment thesignal transduction scheme is dependent of FRET. To be detected,influenza virus particles (neuraminidase tetramers) must bind two ormore receptor molecules. Binding must cause the receptors to aggregateresulting in fluorescence energy transfer. These “receptor molecules”have three functions. First they must have a recognition ligand thatbinds specifically to an agent. The receptor must carry the fluorescentreporter and must be mobile in a lipid bilayer membrane. Diagrarnmed inFIG. 1 is a prototype “receptor”. A trifunctional linker molecule mustconnect the recognition site, fluorescent reporter, and membrane anchor.

[0036] Trifunctional Linker—Because they are available, α-amino acidswith functional groups on the side chains are good candidates astrifunctional linkers. In addition to the α-amino and α-carboxylfunctional groups, common amino acids are available with hydroxyl(serine), carboxyl (glutamate and aspartate), thiol (cysteine) and amino(lysine) functionality in the side chain. Lysine derivatives areavailable with the α-amino and ε-amino groups differentially blocked sotheir chemistry is orthoganal. Influenza receptors are prepared fromcommercial N^(α)-benzyloxycarbonyl-N^(ε)-t-butyloxycarbonyl-L-lysineN-hydroxysuccinimide ester (1, Z-Lys(Boc)-Osu). The N^(α)- andN^(ε)-blocking groups are removed differentially by hydrogenation (CBZ)or dilute acid hydrolysis (Boc). The alkyl anchors are added first bydisplacement of the N-hydroxy-succinimide ester by treatment ofZ-Lys(Boc)-Osu with distearylamine (2, Scheme 1). Next the CBZ group isremoved by hydrogenation under standard conditions. Dialkyl-substitutedamide (4) have been prepared in essentially quantitative yield as thestarting material for the synthesis of the artificial influenzareceptors described here. Recognition ligand is attached through aspacer to the α-amino group. Next the Boc protecting group is removedand the fluorescent probe attached to the ε-amino group.

[0037] The neuraminidase in influenza virus is uniquely a homotetramericprotein. An active site-specific binder of neuraminidase would provide aflu-specific detector using the FRET scheme. Each viral particlecontains several hundred copies of the neuraminidase tetramer. Inaddition, many competitive inhibitors of flu neuraminidase have beendeveloped as anti-viral agents. These inhibitors provide a specificprobe for the active site of neuraminidase. One example,4-acetylamino-3-guanidino benzoic acid (11) binds flu neuraminidase witha 10 micromolar (μm) affinity constant (Sudbeck et. al., Journal ofMolecular Biology, vol. 267, pp. 584-594 (1997)). Derivatives of thesecompounds have been prepared that will be linked to the receptor througheither 4-amino function or the 3-guanidino group. Other nmolarinhibitors are also becoming available from the pharmaceutical industry.

[0038] Among the numerous neuraminidase inhibitors taught by the priorart are thoose compounds described by Luo et al., in U.S. Pat. No.5,453,533, by Bischofberger et al., in U.S. Pat. No. 5,763,483, byBischofberger et al., in U.S. Pat. No. 5,952,375, Bischofberger et al.,in U.S. Pat. No. 5,958,973, Kim et al., in U.S. Pat. No. 5,512,596, Kentet al., in U.S. Pat. No. 5,886,213, Babu et al., in U.S. Pat. No.5,602,277, by von Izstein et al., in U.S. Pat. No. 5,360,817, Lew etal., in U.S. Pat. No. 5,866,601, by Babu et al., in WO97/47194A1, byBabu et al., in WO99/33781A1, and by Brouillette et al., inWO99/14191A1. Each of these various neuraminidase inhibitors may bestructurally incorporated into the influenza sensor of the presentinvention. The various neuraminidase inhibitors taught by theseenumerated patents are incorporated herein by reference.

[0039] Many of the neuraminidase inhibitors having the highest bindingaffinities include at least one functionality from among carboxylate,guanidinium and N-acetyl groups.

[0040] Scheme 2 shows the synthesis of the neuraminidase ligand linkedvia the guanidino group. Commercially available 4-amino benzoic acid (5)is converted to its methyl ester (6) by treatment with methanol/HCl.Such a methylation is described by Haslam, Tetrahedron, 1980, 36, 2409.Methyl 4-amino benzoic acid (6) is treated with acetic anhydride toyield methyl 4-acetylamino benzoic acid (7). Such a treatment isdescribed by Greene, T. W., “Protective Groups in Organic Synthesis”(John Wiley & Sons, New York, 1981), in particular at pages 251-252.Treatment of (7) with one equivalent of nitronium tetrafluoroborate inmethylene chloride yields methyl 3-nitro-4-acetylamino benzoic acid (8).Such a treatment is described by Ottoni et al., Tetrahedron Lett.(1999), 40(6), 1117. Hydrogenation of (8) over Pd/C in ethanol yieldsthe methyl 3-amino-4-acetylamino benzoic acid (9). Such a hydrogenationis described by Entwistle et al., J. Chem. Soc., Perkin Trans 1, 1977,433. Product 9 is alkylated with one equivalent of a spacer moleculewhere n is generally from about 2 to about 6. Such a alklyation isdescribed by Onaka et al., Chem. Lett., 1982, 11, 1783. The secondaryamine is then converted to the guanidino function by treatment withcyanogen bromide followed by ammonia yielding compound 11. Such aconversion is described by Rai et al., Indian J. Chem., Sect. B, 1976,14B(5), 376; by Pankratov et al., Izv. Akad. Nauk SSSR, Ser. Khim. 1975,10, 2198; and by March, “Advanced Organic Chemistry, 4th edition”, (JohnWiley & Sons, New York, 1992), in particular at page 903. A fluorescentgroup is then added to this ligand using the steps below.

[0041] The recognition ligand (11) is then attached via the spacer tothe lipid anchor as outlined in Scheme 3. The free carboxylate on thespacer of compound 11 is attached to the α-amino group of the lysinelinker (4) with dicyclohexyl carbodiimide using standard peptidesynthesis conditions to yield (13). Such a process is described byMarch, “Advanced Organic Chemistry, 4th edition”, (John Wiley & Sons,New York, 1992), in particular at page 420. The methyl ester issaponified from 13 by treatment with lithium hydroxide in THF/water toyield (14). Such a process is described by March, “Advanced OrganicChemistry, 4th edition”, (John Wiley & Sons, New York, 1992), inparticular at page 383. Facile removal of the Boc group from (14) withtrifluoroacetic acid is required before introduction of the fluorescentgroup. Such a process is described by March, “Advanced OrganicChemistry, 4th edition”, (John Wiley & Sons, New York, 1992), inparticular at page 168. The BODIPY fluorophors are available as theirN-hydroxy-succinimide esters. Displacement of the O-Su ester with theE-amino group of the lysine linker will yield the final influenzareceptor (Scheme 4).

[0042] Scheme 4 shows the synthesis of a neuraminidase ligand linked viathe 4-amino group of the 4-acetylamino-3-guanidino benzoic acid-basedinhibitors. Methyl 4-amino benzoic acid (6) is used as the startingmaterial. Treatment of 6 with the acyl chloride 16 yields the amide 17.The 3-amino function is added by treatment of 17 with nitroniumtetrafluoroborate followed by hydrogenation as described in scheme 2.Conversion of the amino function into a guanidino group is accomplishedby treatment with cyanogen bromide followed by ammonia. The terminalhydroxyl on the spacer of compound 20 must be oxidized to a carboxylatefor attachment to the lysine linker. The free carboxylate onamino-linked neuraminidase ligand (21) is attached to the α-amino groupof the lysine linker (4) with dicyclohexyl carbodiimide as described inscheme 3.

[0043] Within the context of the invention samples suspected ofcontaining neuraminidase include natural or man-made materials such asliving organisms; tissue or cell cultures; biological samples such asbiological material samples (blood, serum, urine, cerebrospinal fluid,tears, sputum, saliva, tissue samples, and the like); laboratorysamples; food, water, or air samples; bioproduct samples such asextracts of cells, particularly recombinant cells synthesizing a desiredglycoprotein; and the like. Typically the sample will be suspected ofcontaining an organism which produces neuraminidase, frequently apathogenic organism such as a virus. Samples can be contained in anymedium including water and organic solvent/water mixtures. Samplesinclude living organisms such as humans, and man made materials such ascell cultures.

[0044] In another embodiment, a varied synthesis of the generic linkermolecule can be conducted. As discussed above the signal transductionscheme is dependent on FRET induced by aggregation of two or morefluorescently tagged antibodies bound to a common surface. These“receptor molecules” have three functions. First, they must have arecognition ligand that binds specifically to an agent. For detection ofinfluenza, the recognition ligands can be any neuraminidase inhibitors.In addition, the receptor must carry the fluorescent reporter and mustbe mobile in a lipid bilayer membrane. Diagrammed in FIG. 2 is aprototype “receptor”. Synthetic schemes to prepare the this receptor arediagrammed in schemes 5-6. A trifunctional linker molecule, homoserineconnects the recognition ligand, fluorescent reporter, and membraneanchor. Based on results obtained in developing a cholera sensor, thisprototype receptor has the following design characteristics. It has twoC-18 alkyl chains needed to tightly anchor the receptor in the membrane.The fluorescent reporters are BODIPY-dyes, which are hydrophobic andtethered to the receptor so as to allow the dye to insert into the lipidbilayer. Insertion into the fluid upper leaf of the bilayer shields thedye molecule from non-specific protein-dye interactions, provides longterm stability towards hydrolysis and helps to anchor the antibody tothe membrane. The phosphoryl (PEG)_(n)-spacer will partition into theaqueous phase and has been extensively studied for use as in preparingbio-compatible surfaces. PEG is known to minimize non-specificprotein-surface interactions. (see literature references 20, 23, 27, 41,and 55-57) The length of the spacer can be adjusted by adding more PEGmonomers to optimize fluorescent energy transfer and binding.

[0045] The synthesis of the prototype receptor is outlined in detail inschemes 5 and 6. For each step literature references are included andthe list of references is below. In addition, yields are included forsome steps. Homeserine was chosen as the trifunctional linker because isnot subject to elimination as is serine. Two routes are being exploredto link the spacer to homoserine through either a phospodiester or asulfone. Both of the phosphate and sulfone groups are expected topartition into the aqueous phase. While the phosphodiester linkage ismore similar to phospholipids, the potential advantage of the sulfone isits stability to hydrolysis. A common intermediate in both routes iscompound IV. Commercially available, N-Fmoc O-Trityl homoserine (1) wascoupled to dioctadecylamine (II) using standard peptide couplingconditions to incorporate the membrane anchors (FIG. 2). (see literaturereferences 3, 12, 29, and 53) Removal of the trityl-protecting groupwith trifluoroacetic acid frees the hydroxymethyl group of homoserine(IV) for addition of the spacer. (4-6) In the phosphodiester route,treatment of homeserine (IV) with (tBuO)2P(N(iPr)2) in the presence of

[0046] tetrazole follow by deblocking with trifluoroacetic acid gave thephospho homoserine derivative (V) in an overall yield of 73%. (seeliterature references 33, 37-39, 46, 50, 51, and 54) Standardphosphonate DNA synthesis conditions were used for the condensation ofthe PEG spacer (VI) with the phospo homoserine (V). (see literaturereferences 9, 10, and 40) Oxidation with t-butyl hodroperoxide yieldedthe phosphodiester (VII). (see literature references 2, 17, 21, and 43)The intermediate VII has been prepared, purified and characterized byNIR spectroscopy (overall yield 60%). In the sulfone route, conversionthe hydroxyl group of homoserine to its corresponding bromide (V_(a))was achieved in 73% yield by treatment with triphenyl phosphine andcarbon tetrabromide. (see literature references 26, 28, and 49)Nucleophilic substitution of bromide by a thiol-terminated PEG spacer(VIa) (18, 48) followed by oxidation will give the sulfone (VIIa). (seeliterature references 19, 22, 30, 44, and 52)

[0047] To complete the linker, the terminal amino group on the PEGspacer is freed, a thiol carboxy, amino, or aldehyde reactive group isadded and then the BODIPY dye is added. As diagrammed in scheme 5, thisscheme is depicted only for the phopodiester-linked receptor. Theidentical scheme will be carried out on VIIa to finish thesulfone-linked receptor. First, the BOC-amino protecting group isremoved from VII under acidic conditions. (see literature references4-6) Next reactive group for specific linkage to recognition ligands isadded to the PEG-terminal amino group. For example, a thiol specificdisulfide(see literature reference 11) or maleimide derivative(seeliterature reference 32) is added to react with a free thiol on theneuraminidase inhibitor. Similarly, an aldehyde specific hydrazone isadded to react with the reducing terminal sugar on a sialaic acidcontaining oligosaccharide. (see literature references 16, 31, 35, and36) These reagents,

[0048] diagrammed in FIG. 4, are available as activatedN-hydroxysuccinimide esters (Pierce Chemical Co.) (see literaturereferences 1, 7, 45, and 47), which will react directly with the freeamino group to form an amide linkage. (see literature references 8, 25,and 34) The BODIPY dye is added in “one pot step” involving removal ofthe Fmoc group from the homoserine amino group,(see literaturereferences 13-15) which will be modified with the N-hydroxsuccinimideesters of one of the BODIPY dyes. (see literature references 24 and 42)DCC is dicyclohexylcarbodiimide. HOBT is 1-hydroxybenzotriazole. DEAD isdiethyl azodicarboxylate. TFA is trifluoroacetic acid.

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[0106] Each of the above references is hereby incorporated by reference.

[0107] Although the present invention has been described with referenceto specific details, it is not intended that such details should beregarded as limitations upon the scope of the invention, except as andto the extent that they are included in the accompanying claims.

What is claimed is:
 1. A sensor for the detection of tetramericmultivalent neuraminidase within a sample, where a positive detectionindicates the presence of a target virus within said sample, said sensorcomprising: a surface; recognition molecules situated movably at saidsurface, said recognition molecules capable of binding with saidtetrameric multivalent neuraminidase, said recognition molecules furthercharacterized as including a fluorescence label thereon; and, a meansfor measuring a change in fluorescent properties in response to bindingbetween multiple recognition molecules and said tetramericneuraminidase.
 2. A method of detecting tetrameric multivalentneuraminidase within a sample, where a positive detection indicates thepresence of a target virus within said sample, said method comprising:contacting a sample with a sensor including a surface, recognitionmolecules situated movably upon said surface, said recognition moleculescapable of binding with said tetrameric multivalent neuraminidasewherein said recognition molecules include a fluorescence label thereon;and measuring a change in fluorescent properties in response to bindingbetween multiple recognition molecules and said tetramericneuraminidase.
 3. A sensor for the detection of tetrameric multivalentneuraminidase within a sample, where a positive detection indicates thepresence of a target virus within said sample, said sensor comprising: asurface; at least two different recognition molecules situated movablyupon said surface, said recognition molecules capable of binding withsaid tetrameric multivalent neuraminidase wherein at least onerecognition molecule includes a fluorescence donor label thereon and atleast one recognition molecule includes a fluorescence acceptor labelthereon; and, a means for measuring a change in fluorescent propertiesin response to binding between said two different recognition moleculesand said tetrameric neuraminidase.
 4. A method of detecting tetramericmultivalent neuraminidase within a sample, where a positive detectionindicates the presence of a target virus within said sample, said methodcomprising: contacting a sample with a sensor including a surface, atleast two recognition molecules situated movably upon said surface, saidrecognition molecules capable of binding with said tetramericmultivalent neuraminidase wherein at least one recognition moleculeincludes a fluorescence donor label thereon and at least one recognitionmolecule includes a fluorescence acceptor label thereon; and measuring achange in fluorescent properties in response to binding between said twodifferent recognition molecules and said tetrameric neuraminidase. 5.The sensor of claim 1 wherein said virus is influenza.
 6. The method ofclaim 2 wherein said virus is influenza.
 7. The sensor of claim 3wherein said virus is influenza.
 8. The method of claim 4 wherein saidvirus is influenza.
 9. The sensor of claim 1 wherein said surfaceincludes a fluid membrane thereon.
 10. The sensor of claim 1 whereinsaid recognition molecules include a moiety including a carboxylategroup, an N-acetyl group and a guanidinium group thereon.
 11. Atrifunctional composition of matter comprising: a trifunctional linkermoiety including as groups bonded thereto (a) an alkyl chain adapted forattachment to a substrate, (b) a fluorescent moiety capable ofgenerating a fluorescent signal, and (c) a recognition moiety having aspacer group of a defined length thereon, said recognition moietycapable of binding with tetrameric multivalent neuraminidase.
 12. Thetrifunctional composition of matter of claim 11 wherein said recognitionmoiety includes at least one group selected from a carboxylate group, anN-acetyl group and a guanidinium group thereon.